Calotropis procera extract inhibits prostate cancer through regulation of autophagy

Abstract Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen‐independent PC‐3 and androgen‐sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound‐healing assay and 2′,7′‐dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE‐mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC–MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC‐3 and 22Rv1, in a dose‐ and time‐dependent manner. Also, CPE significantly inhibited the colony‐forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF‐κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE‐treated PC‐3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.


| INTRODUC TI ON
Prostate cancer (PCa) is in fourth position in terms of new incidences and eighth in terms of cancer-related deaths in both sexes with a death rate of 3.8% worldwide.Amongst men, it is the second most common cancer with a global death rate of 6.8% as of 2020. 1 Androgen deprivation therapy (ADT), surgery and radiation therapy are the commonly available primary treatments for PCa patients. 2wever, conventional treatments have several adverse side effects including cardiotoxicity, musculoskeletal alterations and neurotoxicity. 3,4To reduce the side effects, several new therapies such as immunotherapy, complementary and alternative medicines (CAM), vaccines and their combinations are being tested for their efficacy. 5mplementary medicines often include crude plant parts in the form of extracts, powder and tablets. 6These drugs are frequently being used along with surgery, radiotherapy or chemotherapy, either as a replacement of chemical drugs or act in a synergistic manner with other chemical drugs in order to increase the efficacy of the treatment with minimal side effects. 7,8The bioactive compounds naturally found in these plants act against cancer cells by targeting various pro-and anti-oncogenic molecules and signalling pathways. 9erefore, the plant derivatives and their constituent bioactive compounds are being actively tested for their therapeutic potential. 8,10e genus Calotropis belongs to Asclepiadaceae family and consists of only six species. 11Calotropis procera is also known as milkweed, swallowwort, 11 madar and sodom apple. 12It is a tropical shrub found in parts of Africa, Asia and other tropics. 13In India, it is majorly found in wastelands of Assam, West Bengal, Rajasthan and Punjab. 12Different parts of this plant such as seeds, flower, roots, leaves and milky latex are reported to possess insecticidal, analgesic, anti-ulcers, cytotoxic, anti-bacterial, hepatoprotective and cardioprotective properties. 13Different phytochemicals in C. procera latex have shown anticancer potential in both human and animal models, specifically against breast, liver, glioblastoma, colon and PCa. 14,15cently the leaf extract of C. procera has been shown to exhibit anticancer potential against lung 16 and breast cancer 17,18 but its role in PCa has not been explored till date.Thus, the current study investigated the anticancer activity of the leaf extract of C. procera on PCa cells in vitro and its underlying mechanism.

| Preparation of C. procera leaf extract
Plant of C. procera was identified by its flowers, and the leaves were washed and air dried for several days to remove moisture.The leaves were then ground to fine powder in liquid nitrogen.The powdered sample was weighed and methanol (Qualigens, India) was added to it in the ratio of 1:10 weight/volume.The methanolic mixture was shaken for 24 h followed by drying in rotary vacuum evaporator at 50°C.The pellet obtained was dissolved in 80% ethanol at a concentration of 80 mg/mL and stored at 4°C till further use.

| Cell culture
All the cell lines, PC-3, 22Rv1 and RWPE-1, were purchased from the American Type Culture Collection (ATCC) and grown in a 37°C incubator with 5% CO 2 .Androgen-independent PC-3 cells were cultured in F-12K medium while androgen-sensitive 22Rv1 cell line was cultured in RPMI-1640 medium, along with 10% FBS for both cell lines.The normal prostate epithelial cell line, RWPE-1, was cultured in KSFM medium containing 5 ng/mL of recombinant human epidermal growth factor and 0.05 mg/mL of bovine pituitary extract.

| Cell viability assay
MTT assay was used to determine the effect of C. procera leaf extract (CPE) on viability of PC-3, 22Rv1 and RWPE-1 cells. 1900 cells/0.2mL/well were seeded in 96-well culture plates.After 24 h of plating, they were treated with varying doses of CPE (25, 50,   100, 200 and 400 μg/mL) for different time points (24, 48 and 72 h).0.5 mg/mL MTT solution was added to the wells after removing the culture medium, and cells were incubated for 4 h at 37°C, 5% CO 2 .
The MTT solution was then discarded and formazan crystals formed were solubilized in DMSO for 2 h.Absorbance at 570 nm was measured using TECAN plate reader (Mannedorf, Switzerland).The absorbance at 570 nm is directly proportional to the number of viable cells.The percentage cell viability was then calculated as:

| Colony formation assay
To determine the colony formation ability of PCa cells on CPE treatment, colony formation assay was performed as previously described. 20Briefly, the cells were seeded in 6-well plates at different cell densities for vehicle control (100, 250, 500 and Percentage cell viability (CV%) = (Absorbance of treated cells∕Absorbance of control) × 100.1000 cells/well) and treatment group (1000, 2000, 3000, 4000 and 5000 cells/well).After the cells adhered, they were treated with IC₅₀ concentration of CPE (26.5 μg/mL for PC-3 and 20 μg/ mL for 22Rv1) or vehicle control (complete media with ethanol) for 48 h.After 48 h of treatment, the wells were washed and complete fresh media was added.The media was changed after every 3-4 days.When colonies appeared in CPE-treated wells (>50 cells/ colony), the cells were fixed with formaldehyde and stained with crystal violet.The colonies were then visualized and counted under the microscope and plating efficiency (PE) and surviving fraction were calculated as:

| Wound-healing assay
To determine the effect of CPE on the migration of PCa cells, wound-healing assay was performed.For the wound-healing assay, 2.5 × 10 5 cells/well were seeded for PC-3 and 6 × 10 5 cells/well were seeded for 22Rv1 cells in 24-well plates.At 80%-90% cell confluency, a scratch was made passing through the middle region of the well, followed by treatment with either IC₅₀ concentration of CPE or vehicle (complete media with ethanol) taken as control.The width of the scratch was measured at 0 h and 48 h of the treatment.The % wound closure was calculated by the formula: % Wound closure = Width at 0 h − Width at 48 h Width at 0 h × 100 .

| Measurement of reactive oxygen species
The endogenous reactive oxygen species (ROS) levels on CPE treatment, were measured using DCFDA assay.For this assay, 5000 cells/ well were seeded in 96-well plates.Cells were treated with either IC₅₀ concentration of CPE or 500 μM Hydrogen peroxide (H₂O₂) (taken as positive control) for 48 h.After 48 h of treatment, 10 μM of DCFDA was added to each well for 1 h and fluorescence was measured using Tecan multimode microplate reader with excitation and emission at 488 and 525 nm respectively.

| Western blot
The whole cell lysate from cells treated with CPE or vehicle control was isolated using MPER reagent along with inhibitors (1% phosphatase inhibitor, 1% protease inhibitor and 1% PMSF).BCA Assay was performed for measurement of protein concentration in the cell lysates.
Equal amount of protein sample from vehicle control and CPE-treated cells was separated using 12% or 15% SDS-PAGE and then electrotransferred on to a PVDF membrane.The membranes were incubated Caspase 3 activity (Cell Signaling Technology) or GAPDH (Santa Cruz Biotechnology), followed by corresponding HRP conjugated anti-rabbit IgG antibody (Cell Signaling Technology) or anti-mouse IgG antibody (Cell Signaling Technology) incubation for 2 h.After that, the membranes were thoroughly washed in TBST (1×).Finally, the PVDF membrane was probed with ECL solution (Thermo Fisher) and observed in ChemiDoc Imaging System (Azure Biosystems) to visualize the protein bands.

| LC-MS analysis
Liquid chromatography-mass spectrometry (LC-MS) analysis of the leaf extract of C. procera was performed using a high-performance

| Identification of intracellular bioactive compounds
In order to analyse the CPE metabolites entering the cells, the PC-3 and 22Rv1 cells were treated with IC₅₀ concentration of CPE.The cells were trypsinized after 24 h of treatment to give maximum time for the metabolites to enter the cells but not enough time for them to cause cell death.After trypsinization of treated cells, the cell pellet was washed with 1× PBS and centrifuged at 13,000×g for 20 min at 4°C, so as to form a tight pellet.The cell pellet was resuspended in 1 mL of nuclease free water.The cells were then lyophilized.The lyophilized cell lysates were reconstituted in 50% MeOH containing 0.1% FA.The resulting solution was vortexed for 1 min and centrifuged at 10,000 rpm for 10 min at 4°C, and the supernatant was subjected for LC-MS analysis as described above.

| Statistical analysis
Data was analysed using GraphPad Prism, version 8.0 (GraphPad Software Inc).The dose-response curve and IC₅₀ values for each cell line were calculated by non-linear regression analysis.Mean ± SEM was calculated for three independent experiments, each with at least three technical replicates.Two-way anova was used to determine statistical significance for all MTT assays, and unpaired t-test with Welch's correction was used to determine the statistical significance for the results of all other assays and western blots.

| Effect of CPE on cell viability
The effect of CPE on viability of human PCa cells, PC-3 and 22Rv1, was measured by the MTT assay (Figure 1A,B).To measure the effect of CPE on viability of non-cancerous cells, normal prostate epithelial cell line (RWPE-1) was used (Figure 1C).Doxorubicin at a concentration of 100 μM was used as a positive control because it is one of the widely used chemotherapeutic drug for PCa.CPE decreased cell viability of PC-3 and 22Rv1 cells in a dose-and timedependent manner.The IC₅₀ values of CPE for all the three cell lines (PC-3, 22Rv1 and RWPE-1) are given in Table 1.The optimal cytotoxic effect of CPE was observed at 48 h in both PC-3 and 22Rv1 cell lines.Therefore, the PCa cells were treated with IC₅₀ concentration of CPE for 48 h in all further experiments.

| Effects of CPE on cell division ability of PCa cells
To determine the division ability of a single cell to form a colony after treatment with CPE, clonogenic assay was performed.The size and number of colonies formed in each cell line decreased after treatment with CPE as compared to the control.CPE treatment had a more profound effect on cell division capability of PC-3 cells compared with 22Rv1 cells (Figure 2A,B).

| Effect of CPE on cell cycle markers
Since a significant decrease in clonogenic ability of CPE-treated cells was observed in both PCa cells, the protein levels of cell cycle markers, p27 and NF-κB, were checked in both PC-3 and 22Rv1 cells after treatment with CPE (Figure 2C,F).Densitometric analysis of the western blots showed no change in expression of p27 whereas a significant decrease in protein levels of NF-κB (p ≤ 0.05) was observed in CPE-treated PC-3 cells (Figure 2D,E).On the contrary, an increase in p27 expression (p ≤ 0.05) was observed in CPE-treated 22Rv1 cells as compared to vehicle control (Figure 2G).Furthermore, NF-κB expression remained downregulated (p ≤ 0.05), in CPE-treated 22Rv1 cells as well (Figure 2H).

| Effect of CPE on cell migration
Wound-healing assay was performed in order to investigate the changes in the cell migration ability of PCa cells in the presence of CPE (Figure 3A,C).The percentage of wound closure in PC-3 cells treated with CPE was 13.61%, which was significantly less (p < 0.001) than that of vehicle control (99.62%; Figure 3B).In 22Rv1 cells also, the cells treated with IC₅₀ value of CPE had significantly (p < 0.01) lower wound closure of 4.19% compared with that of vehicle control (15.08%) (Figure 3D).

| Effect of CPE on antioxidant markers
As ROS levels decreased after treatment with CPE, the effect of CPE on protein levels of different antioxidant molecules including catalase, superoxide dismutase 1 (SOD1) and thioredoxin were analysed (Figure 4C,G).Though a reduction in catalase levels was observed after treatment of PC-3 cells with CPE, it was not statistically significant (Figure 4D).The levels of SOD1 remained unchanged (Figure 4E) while the levels of thioredoxin decreased significantly (p < 0.01; Figure 4F).

| Effect of CPE on necrosis
Lactate dehydrogenase assay was performed to analyse whether

| Effect of CPE on markers of autophagy
Western blotting for autophagy marker proteins, LC3B, Beclin-1 and p62, was performed to assess whether autophagy is the mechanism involved in decreasing cell viability of PCa cells after CPE treatment (Figure 6A,E).It was observed that in PC-3 cells, a significant increase in the ratio of LC3-II to LC3-I protein levels (p < 0.05) was observed in cells treated with CPE (Figure 6B) but no change in Beclin-1 level was observed between vehicle control and treatment group (Figure 6C).The levels of p62 protein in PC-3 cells were significantly upregulated (p < 0.05) as well (Figure 6D).
However, in 22Rv1 cells, a significant decrease in the ratio of LC3-II to LC3-I and Beclin-1 (p < 0.01) was observed in cells treated with CPE as compared to vehicle control (Figure 6F,G).Though a decrease in p62 levels was also observed in CPE-treated 22Rv1 cells, it was not statistically significant (Figure 6H).

| Effect of CPE on apoptotic markers
In order to investigate whether decreasing cell viability by CPE was BAD was observed while BAX level was significantly downregulated (p < 0.05) in CPE-treated PC-3 cells as compared to vehicle control (Figure 7D,E).Level of the anti-apoptotic protein, Bcl-2, also remained unchanged (Figure 7F).
A similar observation as PC-3 cells was obtained in CPEtreated 22Rv1 cells where BAD protein levels remained unchanged while a significant decrease in BAX level (p < 0.05) was observed (Figure 7H,I).However, a decrease in Bcl-2 levels was also observed, though it was not statistically significant (Figure 7J).

| LC-MS analysis of C. procera leaf extract
A total of 123 bioactive compounds were recognized in positive and negative ionization modes through untargeted LC-MS analysis of CPE.A list of all identified primary and secondary metabolites is given in Table 2.
LC/MS analysis of PC-3 and 22Rv1 cells treated with CPE for 24 h was performed to identify the compounds which get internalized.It was observed that from the 123 metabolites identified in the crude leaf extract, 63 metabolites were be detected in PC-3 cells treated with CPE while they were absent in cell lysates of vehicle control (Table 2).
In 22Rv1 cells, 105 metabolites were identified in the CPE-treated cells which were absent in cell lysate of vehicle control (Table 2).

| DISCUSS ION
Conventional treatment methods for PCa have many side effects and hence, treatment with herbal extracts is considered an important p27kip1 protein directly mitigates inflammation by inhibiting NF-κB activation thus playing a key role between cell cycle and inflammation. 21It is also reported that loss of p27 results in increased aggressiveness of PCa. 22Hence, our observation that p27 increased in CPE-treated 22Rv1 cells may explain our results showing decreased cell division and migration capability of these cells after treatment with CPE.Similarly, downregulated expression of NF-κB in both PCa cell lines after CPE treatment is in line with another study showing that C. procera inhibits breast cancer proliferation by inhibiting NF-κB activation. 23evated ROS levels maintain tumorigenicity and promote genomic instability in cancer cells.A study using various prostate cells showed that ROS generation is higher in PCa cells compared with normal prostate cells and suggested that reducing ROS production may decrease cell migration while increasing cell death. 24terestingly, our data showed that CPE treatment results in significant reduction in ROS levels, which may lead to the reduced cell viability and migration capacity of the PCa cells.Moreover, another study in breast cancer has shown that C. procera can induce cytotoxicity by reducing ROS levels resulting in decreased cell migration and induction of apoptosis. 18duction in ROS levels is usually associated with a concomitant increase in antioxidants. 25Interestingly, our data showed that levels of antioxidant markers, catalase, SOD1 and thioredoxin, decreased after CPE treatment.Since CPE is known to possess significant radical scavenging activity, 16 it can be inferred that the reduction in ROS levels observed in CPE treated PCa cells was direct and not mediated by modulation of antioxidant enzymes.
Also, SOD1 has been proposed as a novel target for anticancer therapy since it remains overexpressed in a number of cancers along with increased ROS levels in order to prevent any damage caused due to excessive ROS and maintain tumorigenesis. 26Similarly, another antioxidant protein thioredoxin showed upregulated expression in androgen-independent PCa and its inhibition led to decrease in cancer growth. 27Thus, it can be proposed that the downregulated protein levels of SOD1 and thioredoxin could contribute to decreasing cell proliferation of PCa cells.
Cancer cells often utilize autophagy for resource reallocation as a survival strategy. 28,291][32] These three proteins play a crucial role in different key steps of autophagy and, hence, were chosen for analysis.In our study, LC3-II and p62 expression was significantly upregulated indicating autophagy induction in CPE-treated PC-3 cells.In contrast, CPE treatment significantly reduced the expression of Beclin-1 and LC3-II in 22Rv1 cells, thus showing inhibition of autophagy.There are data suggesting that depending on the cellular features of the PCa cell lines, either induction or inhibition of autophagy can result in cell survival. 28Studies have shown that activation of autophagy in androgen-independent PC-3 cells results in cell cycle arrest in G2/M phase. 33,34However, it has been observed that autophagy acts as pro-survival mechanism in androgen-responsive 22Rv1 cells. 29me previous studies have shown that C. procera extracts inhibit proliferation of human skin melanoma cells and canine mammary tumour cells via apoptosis induction. 35,36However, our results showed that both extrinsic and intrinsic pathways of apoptosis were
chromatography system (HPLC; Agilent Technologies, Santa Clara, CA, USA) hyphenated to a triple quadrupole tandem mass spectrometer (4000 Q-Trap, AB Sciex, Foster City, CA, USA).All the parameters of mass spectrometer and HPLC were controlled by Analyst software, version 1.7.1 (AB Sciex) and OpenLAB control panel software (Agilent Technologies), respectively.The crude extract was diluted to 1 mg/mL in pure methanol and further diluted to 10 μg/mL in 50% methanol containing 0.1% formic acid (FA) for LC-MS analysis.The chromatographic separation was carried out on a Purospher® STAR RP-18 endcapped (3 μm) column using a mobile phase combination of solvent A (18.2 MΩ Milli-Q water with 0.1% FA) and solvent B (pure methanol with 0.1% FA) pumped at a flow rate of 0.5 mL/min.The gradient profile was set as: 0.0 min 5% B eluent, 1.0 min 5% B eluent, 15.0 min 100% B eluent, 19.0 min 100% B eluent, 20.0 min 5% B eluent and 25.0 min 5% B eluent.The temperature of autosampler tray and the column oven were maintained at 10 ± 1 and 25 ± 1°C, respectively, and the samples were injected at a volume of 20 μL for analysis.In order to achieve maximum coverage of the components, ESI source was operated in dual ionization mode with polarity switching.Independent data acquisition using enhanced mass scan (EMS) mode in the range of 50-800 (m/z) was used for capturing the [M+H + ] and [M-H + ] masses.The source-dependent parameters were set as: curtain gas (CUR) = 25 psi, collision gas (CAD) = 12 psi, ion-spray voltage = 4.5 kV and 5.5 kV for positive and negative mode respectively, source temperature = 500°C, ion source gas 1 (GS1) = 45 psi, ion source gas 2 (GS2) = 60 psi.
DCFDA assay was performed to analyse the effect of CPE on endogenous ROS levels in PCa cells (Figure4A,B).In both PC-3 and 22Rv1, the ROS levels increased after treatment with H₂O₂, which was taken as positive control.Cells treated with vehicle control showed no significant change in ROS levels compared with untreated cells.However, upon treatment with IC₅₀ concentration of CPE, significant decrease in ROS levels was observed in both cell lines compared with untreated cells or compared with cells treated with vehicle control (Figure4A,B).
Interestingly, in CPE-treated 22Rv1 cells, protein levels of catalase and SOD1 were significantly downregulated (p < 0.01), while thioredoxin expression remained unchanged, as compared to vehicle control (Figure4H-J).
CPE induces necrosis inPCa cells (Figure 5A,B).The percentage cell cytotoxicity was significantly reduced in both PC-3 (p < 0.01) and 22Rv1 (p < 0.05) cells on treatment with CPE indicating a reduction in necrosis as compared to vehicle control (Figure 5A,B).F I G U R E 1 Effect of Calotropis procera leaf extract (CPE) on cell viability: Per cent cell viability at different time points (24, 48, 72 h) after treatment with varying concentrations of CPE (25-400 μg/ mL).(A) PC-3 cell line (B) 22Rv1 cell line (C) RWPE-1 cell line.Data are presented as mean ± SEM (n = 3).
Caspase activity assay was performed to analyse whether CPE reduces the cell viability of PCa cells by inducing apoptosis through extrinsic pathway.No significant change in caspase activity was observed in CPE-treated cells as compared to vehicle control in both PC-3 and 22Rv1 cells (Figure 7A,B).

F I G U R E 2
mediated through intrinsic apoptotic pathway, protein expression of apoptotic markers such as BAD, BAX and Bcl-2 was measured in cells treated with CPE (Figure 7C,G).No change in protein level of Effect of Calotropis procera leaf extract (CPE) on cell division ability measured via clonogenic assay and western blot of cell cycle markers in prostate cancer cell lines.(A, B) Histogram represents the survival fraction of the colonies formed in (A) PC-3 and (B) 22Rv1 cells treated with CPE or vehicle control.(C-E) Representative western blot and densitometric analysis of cell cycle markers p27 and NF-κB in PC-3 cells.(F-H) Representative western blot and densitometric analysis of cell cycle markers p27 and NF-κB in 22Rv1 cells.Data are represented as mean ± SEM (n = 3).*p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle control.

F I G U R E 3
Effect of Calotropis procera leaf extract (CPE) on cell migration of prostate cancer cells after 48 h of treatment.Representative images showing the migration of cells in the wound area in (A) PC-3 and (C) 22Rv1 cells.Percentage of wound closure in (B) PC-3 and (D) 22Rv1 cells treated with CPE or vehicle control.Data are represented as mean ± SEM (n = 3).**p < 0.01, ***p < 0.001 compared with vehicle control.

F I G U R E 4
Levels of endogenous reactive oxygen species (ROS) measured by DCFDA assay and protein levels of antioxidants markers in prostate cancer cells treated with Calotropis procera leaf extract (CPE).Endogenous ROS levels after CPE treatment in (A) PC-3 cells (B) 22Rv1 cells.H₂O₂ was used as positive control in this assay.(C-F) Representative western blot and densitometric analysis of antioxidant markers catalase, SOD1 and thioredoxin in PC-3 cells.(G-J) Representative western blot and densitometric analysis of antioxidant markers catalase, SOD1 and thioredoxin in 22Rv1 cells.The data are shown as mean ± SEM (n = 3).ns , no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001 compared with vehicle control or only cells.form of alternative therapy.Thus, in this study we investigated the effect of the leaf extract of the herb, C. procera on PCa cell lines in vitro.Results showed that leaf extract of C. procera significantly decreased cell viability as well as cell division and migration capability of both androgen-independent and androgen-sensitive PCa cell lines.Interestingly, the androgen-sensitive 22Rv1 cell line was more susceptible to CPE treatment as compared to androgen-independent PC-3 cell line as indicated by the IC 50 values.

F I G U R E 5 41 F I G U R E 6
not activated in PCa cells by CPE treatment.Decrease in cell viability by CPE also did not involve necrosis as shown by the results of LDH Release assay.In order to identify the compounds of C. procera hydroalcoholic extract which may be inhibiting cell viability of PCa cells, LC-MS analysis of the hydroalcoholic extract as well as the cells treated with CPE was performed.Majority of compounds internalized by both PCa cell lines belonged to the class of terpene derivatives, cardenolides and flavonoid glycosides.Interestingly, more of the compounds were internalized by the androgen-responsive 22Rv1 cells with only three compounds, namely nicotiflorin, kaempferol robinoside and α-cyclotral internalized specifically by androgen-independent PC-3 cells.Terpenes like lupeol have been known to suppress tumour angiogenesis via downregulation of TNFα. 37Another terpene Effect of Calotropis procera leaf extract (CPE) on necrosis of (A) PC-3 cells and (B) 22Rv1 cells as analysed by the lactate dehydrogenase release assay.Doxorubicin was used as positive control in this assay.The data are shown as mean ± SEM (n = 3).ns , no significant difference; *p < 0.05, **p < 0.01, ***p < 0.001 in CPE-treated cells compared with vehicle control cells or doxorubicintreated cells.derivative hinesol has shown anti-proliferative effect in lung cancer cells. 38Cardenolides like uscharin, calotropin, calactin, 2′-oxovoruscharin, 19-dihydrocalactin, 19-dihydrocalotoxin, 15-β hydroxy uscharin, asclepin and calotropagenin have been known to inhibit Na+/K+ATPase and HIF-1α activity, which are important for tumour metastasis.Thus, these cardenolides show cytotoxic effect against breast, colon, cervical, lung and ovarian cancer. 39-Effect of Calotropis procera leaf extract (CPE) on molecular markers of autophagy in PC-3 and 22Rv1 cells.(A-D) Representative western blot and densitometric analysis of autophagy markers in PC-3 cells.(E-H) Representative western blot and densitometric analysis of autophagy markers in 22Rv1 cells.Data are represented as mean ± SEM (n = 3).*p < 0.05, **p < 0.01 between vehicle control and CPE-treated groups.F I G U R E 7 Effect of Calotropis procera leaf extract (CPE) on induction of apoptosis, analysed by caspase activity assay and western blot of apoptotic markers.Caspase activity after CPE treatment in (A) PC-3 cells and (B) 22Rv1 cells.(C-F) Representative western blot and densitometric analysis of apoptotic markers BAD, BAX and Bcl-2 in PC-3 cells.(G-J) Representative western blot and densitometric analysis of apoptotic markers BAD, BAX and Bcl-2 in 22Rv1 cells.The data are shown as mean ± SEM (n = 3).ns = no significant difference, *p < 0.05 in CPE-treated cells compared with vehicle control cells.

| Measurement of apoptotic potential
Compounds identified in Calotropis procera leaf extract.